1. Field of the Invention
The present invention relates generally to the field of immunoassays, and more particularly to an immunoassay intended to measure the cellular proteins from a host cell grown in cell culture. The host cell will generally have been used to produce a protein which has been isolated and purified for therapeutic use. The therapeutic protein will generally have been engineered into the host cell by recombinant DNA techniques.
2. Description of the Related Art
Immunoassays are generally known in the art. These assays use an antibody or antibodies to measure the amount of a given antigen in a sample. A typical immunoassay format, known generally as a sandwich ELISA (enzyme-linked immunosorbent assay), uses an enzyme-linked antibody for detection purposes. In one format, this detection antibody binds to an antigen to be assayed. First a "capture" antibody is immobilized, such as by coating it onto the well of a plastic plate. A sample containing an unknown amount of antigen is then applied. The antigen is captured by the antibody on the plate. The plate is washed free of other proteins and other materials. Then, the detection antibody is applied, and it binds to the antigen captured by the capture antibody. This detection antibody has been coupled to a chemical marker, typically an enzyme, which gives off a color when reacted with a substrate. Alternatively the detection antibody can be coupled with biotin, and the antibody detected using enzyme-conjugated streptavidin or avidin. The amount of detection antibody bound to the complex is then determined colorimetrically.
This format of ELISA is termed a "sandwich" ELISA, in that the antigen is akin to a slice of meat between two slices of antibody bread. An assay in this format usually uses the same polyclonal antisera for the capture antibody and the detection antibody. Monoclonal antibodies are only used for both capture and detection antibodies when they have been found to be reactive with different sites on the antigen.
A review of various enzyme immunoassays is contained in Kurstak, "Progress in Enzyme Immunoassays, Experimental Design, and Interpretation," Bull. W.H.O. 63(4):793-811 (1985). Because of its description of various immunoassay formats which may be used in connection with the present invention, this disclosure is hereby incorporated by reference into the present specification. A specific example of an immunoassay used in the production of a recombinant DNA product is found in Ferrua et al., "Human Interleukin 2 Detection at the Picomolar Level by Sandwich Enzyme Immunoassay," J. Immunol. Methods, 97:215-220 (1987).
Anicetti et al., "Immunoassay for the Detection of E. Coli Proteins in Recombinant DNA Derived Human Growth Hormone," J. Immunol. Methods, 91:213-224 (1986), describe an ELISA test for the quantitative measurement of E. coli host cell proteins in recombinant human growth hormone. This is a multiple antigen immunoassay using affinity purified polyclonal antibodies. Because E. coli is not grown in media containing proteins and is processed from a cell paste, there is no suggestion in this article that one should or could deplete the antibody of anti-media antibodies. In the series of experiments described in this reference, the antiserum is purified by passing it over a chromatography column containing reference E. coli proteins. Only the antibodies which bound to the immobilized E. coli proteins were used in the assay.
Lucas et al., "Enzyme-linked immunosorbent assays (ELISAs) for the determination of contaminants resulting from the immunoaffinity purification of recombinant proteins," J. Immunol. Methods, 13:113-122 (1988) disclose ELISA's for the determination of contaminating proteins in rDNA and monoclonal antibody products produced in mammalian cell culture. However, these assays are direct assays for one particular contaminating protein, e.g. the bovine IgG contained in fetal bovine serum. A specific antibody to this IgG is used.
Depleted antisera have been previously used in certain test procedures. Lundblad et al., "The Antigenic Nature of Heat Treated Human Plasma Proteins," Vox. Sang. 5:122-137 (1960) describe in a general sense the use of a depleted antiserum to evaluate the presence or absence of a particular antigen in an Ouchterlony assay.
J. C. Giddings, "The Purification of Factors VIII and IX, and Production of Specific Antisera," Vol. 5 Ch. 4, Methods in Haematology, 1982, discloses absorption of antisera to F.VIII:RAg with F.VIII:RAg-deficient plasma. This was to produce an antiserum specific to F.VIII:RAg.
Other assays have previously employed depleted anisera. See, e.g. Venn et al., "Limitations of a hemolytic plague assay for IgG-anti-IgG rheumatoid factor-producing cells." J. Immunol. Methods 102:195-204 1987.